The long term goal of this project is to understand the mechanisms that govern receptor uptake and transport within cells since failure to internalize cell surface receptors can result in heart disease, leukemia, and breast cancer. The clathrin-mediated endocytic pathway is the major mechanism for receptor internalization in eukaryotes. Clathrin assembly at the plasma membrane drives receptor uptake. However, its coupling to receptors and other endocytic components requires adaptor proteins like ARH, Dab2, and Numb. These proteins are PTB (phosphotyrosine binding) domain-containing endocytic adaptors that control the spatio-temporal organization of the receptor transport machinery. They are modular with distinct protein interaction platforms that enable them to promote assembly of protein complexes. While genetic and cell biological studies reveal the importance of PTB adaptors, the mechanisms that regulate their dynamic activities in receptor transport remain unknown. For example, what controls endocytic machinery assembly at the appropriate time and place? What mechanisms regulate cargo recognition and adaptor protein interaction with other endocytic components? Experiments in this proposal will begin to address these unanswered questions. Our preliminary observations support the idea that adaptor protein phosphorylation controls receptor transport. Indeed the AP2-associated kinase, AAK1L, performs essential roles in multiple transport steps by targeting different endocytic adaptors including AP2, Numb, and ARH. We postulate the AAK1L modulates the dynamic scaffolding activities of adaptors to influence where and when the endocytic machinery is assembled. In this application, we will pursue three specific aims to test this hypothesis. 1) We will resolve the molecular basis for AAK1L action in receptor recycling. This will be accomplished using a rescue approach for transferrin receptor (TfnR) recycling in AAK1L-depleted cells. We will also use biochemical interaction screens to identify AAK1L endosomal substrates. 2) We will determine how phosphorylation controls Numb activity in coated pit assembly with in vitro binding assays to test the consequence of phosphorylation on Numb interaction with Eps15 and AP2. Additionally, quantitative internalization assays will be combined with immunolocalization experiments to dissect how Numb phosphomutants disrupt receptor endocytosis. 3) We will define how AAK1-mediated ARH phosphorylation regulates low density lipoprotein receptor (LDLR) endocytosis using in vitro assays to determine if ARH phosphorylation impacts binding to the LDLR or the core endocytic machinery. In vitro studies will be validated with in vivo experiments where we will analyze the role of ARH phosphorylation in LDLR clustering and recruitment to clathrin-coated pits. Collectively, results from these studies will not only provide important mechanistic insight into how transport efficiency and fidelity is maintained for nutrient receptors like LDLR and TfnR, but they may also serve as a paradigm for understanding the mechanisms that control the down-regulation of signaling receptors. Public Health Relevance: This project seeks to better define how the liver regulates cholesterol removal from the blood by understanding the key events that control the uptake machinery.